The concept of synthetic lethality, or conditional genetics, describes the genetic interaction of two genes, both involved in a cellular process. When either gene is mutated alone, the cell remains viable. However, the combination of mutations in these two genes results in cell death (Hartwell, et al., Science, 278:1064-1068 (1997)). In the case of chemical synthetic lethality, the first mutation is essential to the development of cancer, while a second gene is inhibited by a small molecule, resulting in cytotoxic cell death (Kaelin, W. G., Jr., Nat Rev Cancer, 5:689-698 (2005); Sutphin, et al., Cancer Res., 67:5896-5905 (2007)). This approach is particularly attractive because it should not affect normal, non-cancerous tissue. Furthermore, synthetic lethality is a therapeutically advantageous approach to drug discovery and is particularly suited to developing therapeutics to treat cancers. It describes a genetic interaction whereby the combination of mutation and/or inhibition of two genes leads to tumor cell death. If only one of these two genes is altered, there are no deleterious effects. For example, in the vast majority of renal carcinomas, the VHL tumor suppressor gene is inactivated, driving growth and expansion.
Conventional chemotherapeutic agents have been identified only by their ability to kill rapidly proliferating cells and therefore such agents cannot distinguish between normal, healthy dividing cells and tumor cells. For this reason, standard agents have low therapeutic indices and are often limited by their severe toxicity to normal tissue. While many solid tumors respond to different combinations of cytotoxic chemotherapies, kidney cancer is a particularly intractable disease. Renal cell carcinoma (RCC), the most common type of kidney cancer, has proven to be particularly challenging, resistant to both radiation therapy and standard systemic chemotherapies (Atkins, et al., Clin Cancer Res., 10:6277 S-6281 S. (2004); Motzer, R. J., and Russo, P., J Urol., 163:408-417 (2000)). To date, immunotherapy using interferon or interleukin-2 has had mild success with responses in less than 10% of patients with metastatic RCC (Rosenberg, et al., Ann Surg., 228:307-319 (1998)). The recent development of anti-angiogenic therapies sunitinib (Sutent) and sorafenib (Nexavar) is encouraging although these agents are not curative (Ahmad, T., and Eisen, T., Clin Cancer Res., 10:6388 S-6392S (2004); Motzer, et al., J Clin Oncol., 24:16-24 (2006)). The targeting of receptor tyrosine kinases, which is not specific to the development of RCC, has become the standard of care for advanced RCC (Rathmell, et al., Curr Opin Oncol., 19:234-240 (2007)). One key distinguishing feature in RCC is the loss of function of the VHL tumor suppressor gene, an essential and frequent mutation in the development of RCC. In order to specifically target RCC cells without toxicity to normal cells, we have employed a synthetic lethal approach, seeking to identify compounds that exhibit selective cytotoxicity to cells that have lost functional VHL.
Tumor hypoxia has a well defined role in driving tumor progression and metastasis, as well as resistance to therapy. A key mediator of hypoxic stress is HIFα. HIF is a bHLH heterodimeric transcription factor, made up of an oxygen-labile subunit (HIF-α) and a constitutive subunit (HIF-β). In the presence of oxygen, hydroxylation on proline residues 564 and 402 by prolyl hydroxylases (PHDs) marks HIF-α for recognition and binding with Von Hippel-Lindau protein (pVHL), leading to degradation of HIF-α. Under hypoxic conditions, activity of the PHDs decrease, which prevents the recognition of HIF-α by pVHL. In cells that lack VHL, stabilized HIF-α binds HIF-β to activate the transcription of genes involved in several processes. HIF transcribes genes that mediate glycolysis, angiogenesis, tissue remodelling, epithelial permeability and vascular tone. These genes, and processes driven by these genes, act to promote tumor growth and survival in hypoxic conditions.
Functional studies indicate that pVHL, the protein product of VHL, is an E3 ubiquitin ligase that targets the α-subunit of the hypoxia-inducible factor (HIF) for proteasomal degradation under normoxia. In addition to its role in HIF regulation, pVHL has been implicated in a variety of processes including extracellular matrix assembly, regulation of microtubule stability, polyubiquitination of atypical PKC family members, regulation of fibronectin, and RNA polymerase II subunits. Glucose transporter 1 (GLUT1), also known as solute carrier family 2 (SLCA2) or facilitated glucose transporter member 1 (SLC2A1) is a 492 amino acid protein (NCBI accession numbers NP—006507.2 or P11166.2). GLUT1 is a member of a small family 45-55 kDa hexose transport proteins and is invovled in facilitating the transport of glucose across the plasma membranes of mammalian cells. (See, e.g., Doege et al., Biochem J., 15:(359):443-449 (2001); Mueckler, et al., Science 229(4717):941-945 (1985); and Olsen et al., Annual Review of Nutrition, 16:235-256 (1996)).
There is considerable interest in the identification of inhibitors of HIF and its downstream genes such as GLUT1. A variety of pharmacological HIF inhibitors have been identified, although the interaction of these agents is not directly with HIF, but via modulation of cellular processes in which HIF is integral.
An extension of this therapy would be in the treatment of cells defective in the von Hippel-Lindau gene and diseases associated with such defects or inhibition of downstream pathways such as inhibition of GLUT1 activity.
Identifying new molecular targeted therapies that specifically kill tumor cells while sparing normal tissue is the next major challenge of cancer research. A characteristic of VHL-deficient cells, namely reliance on GLUT1 and aerobic glycolysis can now be exploited in treatment of diseases related to rapidly dividing cells. High-throughput chemical synthetic lethal screens have been used to identifiy small molecules that exploit the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in approximately 80% of renal carcinomas. These small molecules selectively kill cells with mutant VHL but not cells with wild-type VHL by specifically targeting glucose uptake via GLUT1 in VHL-deficient tumors, which are dependent on glycolysis for ATP production. The present application describes small molecules that impair glucose transport in VHL-deficient cells, but not in cells with wild-type VHL, resulting in specific killing of renal carcinoma cells. The potential to target glucose uptake in VHL-deficient tumors therapeutically with the use of small molecules provides a new way to treat metastatic renal carcinoma, among others types of diseases mediated by elevated expression of GLUT1. Treatment with these small molecules inhibits the growth of VHL-deficient tumors by binding GLUT1 directly and impeding glucose uptake in vivo without toxicity to normal tissue.